BACTERIAL COMMUNITY PROFILING OF WESTERN AUSTRALIAN BOBTAIL LIZARD (TILIQUA RUGOSA) TICKS Ruby Mckenna Bachelor of Science Submitted in fulfilment of the requirements for the degree of Honours in Biological Sciences School of Veterinary and Life Sciences Murdoch University, Perth 2019 iii Statement of Original Authorship I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. Ruby McKenna iv Abstract Ticks are haematophagous arthropods and major vectors of pathogenic microorganisms. In Australia, over 74 species of ticks have been described, of which 13 are known to parasitise reptiles. While only three tick borne diseases are formally recognised, Rickettsia honei, the causative agent of Flinders Island spotted fever, has been long associated with the reptile tick, Bothriocroton hydrosauri, despite this tick rarely reported to parasitise people. More recently, a novel Rickettsia species, Rickettsia gravesii, was reported in the ornate kangaroo tick (a common human biting tick), Amblyomma triguttatum, on Barrow Island in Western Australia. In addition, a number of overseas tick-associated microbes (taxa of interest) have been identified in Australian human-biting ticks using an advanced molecular technique, next generation sequencing (NGS). Therefore, the aims of this study were to morphologically and molecularly identify ticks that parasitise reptiles, specifically the bobtail lizard, Tiliqua rugosa, and to employ NGS to profile the bacterial 16S rRNA gene (16S) within the ticks. The taxa identified would then be phylogenetically compared to known taxa of interest. A total of 306 ticks from Western Australia were morphologically identified and 30 were removed from the data set and the remaining 276 included all developmental stages comprising males 43.1% (n=119); nymphs 40.6% (n=112); females 15.2% (n=42) and three larvae (1.1%). Using Australian-specific morphological keys, ticks were identified as A. albolimbatum. To provide more accurate species identification, molecular barcoding of the cytochrome C oxidase 1 (CO1) gene was employed on 17 nymphal and two larval ticks, along with Northern and Southern WA A. albolimbatum ticks as representative specimens. However, only one nymph (5.8%) and two larvae (100%) generated clean chromatograms and all were identified as A. albolimbatum; the two larvae were genetically identical to the Southern representative sample and the nymph more genetically similar to the Northern representative iii sample (0.48% genetic difference). The genetic distance between the two A. albolimbatum sequences was 4.79%. A total of 116 ticks and six controls were processed for 16S metabarcoding to profile the bacterial communities. A total of 9,706,920 reads were generated using an Illumina V3 600 cycle run on the MiSeq platform. A final quality filtered data set consisted of 4,823,227 reads, with a total of 1,385 zero operational taxonomic units (ZOTUs) generated. The bacterial diversity of the ticks was observed to be statistically different between life stage, with males exhibiting the highest diversity. The bacterial microbiome of the tick samples was dominated by the phylum Proteobacteria (90.94%) and also included Actinobacteria (3.81%) and Firmicutes (3.71%). Interestingly, orders Rickettsiales and Legionellales, which contain known taxa of interest, were identified. BLAST analysis of ZOTUs associated with taxa of interest revealed the most abundant ZOTU as Rickettsia endosymbiont (100% similarity; GenBank accession MK00580); ZOTU2 was 99% genetically similar to Francisella hispaniensis (GenBank accession KT28184); while ZOTU10 and ZOTU19 were most closely related to a Spotted Fever Rickettsia raoultii (99.7% similarity; GenBank accession MK30454) and Coxiella burnetii (100% similarity; GenBank accession LC464975), respectively. Importantly, while R. raoultii does not exist in Australia, literature supports that species delimitation for Rickettsia cannot be based on 16S alone and requires a multi-loci approach. Therefore, the Rickettsia identified in this study may in fact represent a novel endemic species. However, the presence of C. burnetii is intriguing and represents the first evidence within A. albolimbatum ticks supported by phylogenetic analysis. While ticks are rarely implemented in the zoonotic transmission of C burnetii, further research is required to investigate the vector competency of this tick and to determine if reptiles, particularly T. rugosa, are viable reservoirs for this bacterium and determine if reptiles pose a risk to wildlife carers. Overall, this study provides new molecular data for A. albolimbatum and requires further research to investigate the validity of the Northern and Southern morphotypes identified. iv Furthermore, for the first time in Australia, this study presents the bacterial communities within the bobtail tick, A. albolimbatum, and showcases a rich diversity of microbes, including endosymbionts and known tick-associated pathogens. v Acknowledgements Firstly, thank you to my supervisors, Dr Charlotte Oskam (Primary) and Professor Peter Irwin (Co-supervisor). Charlotte, thank you for your generosity, inspiring ideas and approachable nature. Thank you to the Vector and Waterborne Pathogens Research Group you have been wonderful. A special thanks goes to to Dr Jill Austen, Megan Evans and Siobhon Egan. Thank you to Siobhon, for your mentorship throughout my project. You have exceptionally generous with your time and patience. I would also like to thank Samuel Bollard, Aimee Carpenter, Wenna Cung for your support throughout this honours year. Thank you to the many people who contributed ticks to this project and especially to Kanyana Wildlife Centre for being so helpful with their patient information. I would also like to thank Gerrut Norval of Flinders University for his helpful tick identification information. I would like to thank The Harry Butler Institute, The Myrtle AB Lamb Scholarship and the Loneragan Family Scholarship for their interest in my research, and their generosity in awarding me scholarships. Finally, thank you to my family and especially my husband Daniel, you are an amazing support. vi Table of Contents Statement of Original Authorship ............................................................................................ iv Abstract .................................................................................................................................... iii Acknowledgements.................................................................................................................. vi Table of Contents .................................................................................................................... vii List of Figures ........................................................................................................................ viii List of Tables ........................................................................................................................... ix List of Abbreviations ................................................................................................................ x Chapter 1: Introduction ......................................................................................................... 1 1.1 Tiliqua rugosa (Bobtail) ................................................................................................. 1 1.2 Ticks ............................................................................................................................... 2 1.3 The tick microbiome and tickborne disease ................................................................... 9 1.4 Characterisation of the tick microbiome ...................................................................... 11 1.5 Microbes Associated with Bobtails ............................................................................. 17 Chapter 2: Materials and Methods ............................................................................ 21 Chapter 3: Results ........................................................................................................ 32 3.1 Taxonomic Identification of Tiliqua rugosa ticks ........................................................ 32 3.2 Next Generation Sequencing data exploration ............................................................. 44 Chapter 4: Discussion .................................................................................................. 63 4.1 Tick identification ........................................................................................................ 63 4.2 NGS data exploration. .................................................................................................. 66 4.3 Conclusions .................................................................................................................. 76 References .............................................................................................................................. 79 Appendices ............................................................................................................................ 87 vii List of Figures Figure 1.1 Phylogeny of the Families, subfamilies and genera of the suborder Ixodida 4 Figure 3.1 Geographic distribution of ticks collected for this study 26 Figure 3.2 Amblyomma nymphal ticks with varying degrees of engorgement 27 Figure 3.3 Female Amblyomma